Prostate apoptosis response-4 protein (Par-4; also known as PAWR) is a leucine zipper domain protein that is conserved in vertebrates. During the previous funding period, we showed the core effector domain of Par-4 (amino acids 137-195, designated the SAC domain) is necessary and sufficient to induce apoptosis. Interestingly, both Par-4 and its SAC domain induce apoptosis selectively in cancer cells, but not in normal cells. Although ectopic Par-4 and the SAC domain have long been thought to effect apoptosis by acting within the cell nucleus to inhibit NF-kappaB, our recent transient transfection studies unexpectedly displayed apoptosis in neighboring cells that did not express the Par-4 or SAC plasmid. Moreover, conditioned medium (CM) from Par-4 transfected cells induced apoptosis when applied to cancer cells, but not to normal cells; this implies over-expression of Par-4 leads to the secretion of a pro-apoptotic activity. Both Par-4 and the SAC protein were detected in the CM by Western blot analysis, and the apoptotic activity in this CM was neutralized by antibodies against Par-4. The apoptotic activity was diminished in the CM from Par-4 transfectants treated with brefeldin A, indicating Par-4 and SAC secretion (or sPar-4 and sSAC, respectively) occurs by the classical pathway (i.e., from the endoplasmic reticulum to the Golgi, and then to the cell membrane). Importantly, CM from normal and cancer cells contains endogenous Par-4, of which the levels increase upon treatment with TRAIL (a short term exposure that is insufficient to produce apoptosis), thereby indicating secretion is physiologically relevant, and not an artifact associated with over-expression of the Par-4 and SAC constructs. Based on these data, we hypothesize endogenous and exogenous sPar-4 and sSAC exert cancer-specific apoptotic effects via both intracellular and extracellular mechanisms. We will address this hypothesis from a mechanistic perspective, characterizing secreted Par-4 and SAC in prostate cancer cells, and clarifying the means by which they selectively initiate apoptosis in cancer cells. Specifically, we will 1) Delineate the mechanism of Par-4/SAC secretion and apoptosis by sPar-4/sSac in prostate cancer cells; 2) Test the growth suppressive effects of sPar-4 and sSAC domain in prostate cancer models; and 3) Study the inter-dependence of extracellular and intracellular Par-4 in inducing apoptosis in cancer cells. As our recent studies indicate Par- 4 and its SAC domain are secreted, and that recombinant (r) Par-4 or rSAC can induce apoptosis when applied exogenously to cells, these data may hold significant clinical relevance. Our results introduce the novel prospect of using sPar-4 or sSAC to induce apoptosis, not only in local or superficially confined tumors into which Par-4 or SAC can be readily delivered intratumorally, but also to distant metastases by intravenous injection or bone-marrow transplantation, which we will explore in this proposal. PUBLIC HEALTH RELEVANCE: Our research focuses on the secreted forms of Par-4 and it core effector domain SAC, both of which selectively induce apoptosis in cancer cells. We will study these two proteins from a mechanistic perspective both in vitro and in vivo, characterizing secreted Par-4 and SAC in prostate cancer cells, and clarifying the means by which they selectively initiate apoptosis in cancer cells. The proposed science will explore the novel prospect of using sPar-4 or sSAC to induce apoptosis, not only in local or superficially confined tumors into which Par-4 or SAC can be readily delivered intratumorally, but also to distant metastases by intravenous injection or bone-marrow transplantation.